Evidence exists in the literature and from our laboratories to implicate PKC in the mechanism of morphine tolerance. It is clear that its role varies with different levels and durations of morphine tolerance. Its role in tolerance to other opioids has not been studied in detail. The importance of PKC in relation to other neuronal constituents has not been well defined. The overall goal of the work proposed in this application is to carry out a collaborative, comprehensive investigation involving electrophysiological techniques at the cellular level (University of Bristol) with whole animal studies (VCU) to elucidate not only if but also how PKC plays a pivotal role in morphine and other opioid tolerance. Another important goal of the research is to elucidate the relative importance of PKC to other cellular proteins such as PKA, arrestins, GRK etc in opioid tolerance. We propose to study the specific isoforms of PKC and to use knockout mice and siRNA techniques in both laboratories to elucidate the mechanisms involved. Close and continual collaboration between the two groups will provide a mechanism for daily input (email and data transfer) into the design of coordinated experiments in each laboratory. This immediate interchange of result will directly affect the design of the next experiment in the other laboratory working at a different level of integration. The specific aims of the project are to test the hypotheses that (1) PKC plays the predominant role in acute moderate morphine tolerance, and that PKA along with PKC are involved in chronic higher levels of morphine tolerance,(2) that opioids with a higher efficacy at mu opioid receptors produce a non-PKC dependent desensitization at MORs and tolerance to these agents in the whole animal is less easily reversed by PKC inhibitors, (3) GRKs, phosphatases and Gq-coupled receptors play a role in some opioid tolerances and (4) that presynaptic sites contribute to both MOR desensitization and opioid tolerance in mice. The relevance of this research to public health is appreciated when one considers that tolerance to opioids limits their use in treating patients such as those with cancer who have chronic pain. We need to understand the mechanisms of tolerance if we are going to alleviate this problem. Also it is essential that we elucidate the mechanisms of tolerance and dependence if we are going to solve the enormous health problems caused by drug abuse.